Effect of Intracellular pH on the Torque–Speed Relationship of Bacterial Proton-Driven Flagellar Motor

Nakamura, Shuichi; Kami-ike, Nobunori; Yokota, Jun; Kudo, Satoshi; Minamino, Tetsuo; Namba, Keiichi

doi:10.1016/j.jmb.2008.12.034

Cited by 73 publications

(93 citation statements)

References 33 publications

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“…It has been shown, however, that the motor speed near zero load is independent of the stator number (26,27). To reduce the rotation rate further under low load, we reduced the intracellular pH without changing the proton motive force (pmf) significantly by lowering the external pH in the presence of 20 mM potassium benzoate (28,29). We carried out bead assays at an external pH of 6.0 in the presence of 20 mM potassium benzoate and 25 μM IPTG.…”

Section: Resultsmentioning

confidence: 99%

“…Salmonella strains SJW46 (fliC(Δ204-292)) (37) and MM3076iC (Δ(cheA-cheZ), fliC (Δ204-292)) (29) were used in this study. SJW46 has intact motors and chemotaxis system, and hence the motor can rotate both CCW and CW, while MM3076iC has intact motors but lacks the chemotaxis system, and hence the motor rotates exclusively CCW.…”

Section: Methodsmentioning

confidence: 99%

“…Bead assays with 1.0 μm polystyrene beads (Invitrogen) and 0.1 μm fluorescent beads were carried out as previously described (14,29).…”

Section: Methodsmentioning

confidence: 99%

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Evidence for symmetry in the elementary process of bidirectional torque generation by the bacterial flagellar motor

Nakamura

Kami-ike

Yokota

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The bacterial flagellar motor can rotate in both counterclockwise (CCW) and clockwise (CW) directions. It has been shown that the sodium ion-driven chimeric flagellar motor rotates with 26 steps per revolution, which corresponds to the number of FliG subunits that form part of the rotor ring, but the size of the backward step is smaller than the forward one. Here we report that the protondriven flagellar motor of Salmonella also rotates with 26 steps per revolution but symmetrical in both CCW and CW directions with occasional smaller backward steps in both directions. Occasional shift in the stepping positions is also observed, suggesting the frequent exchange of stators in one of the 11-12 possible anchoring positions around the rotor. These observations indicate that the elementary process of torque generation by the cyclic association/ dissociation of the stator with every FliG subunit along the circumference of the rotor is symmetric in CCW and CW rotation even though the structure of FliG is highly asymmetric and suggests a 180°rotation of a FliG domain for the rotor-stator interaction to reverse the direction of rotation.FliG | MotAB stator complex | Salmonella | stepping rotation | switch

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Evidence for symmetry in the elementary process of bidirectional torque generation by the bacterial flagellar motor

Nakamura

Kami-ike

Yokota

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…It has been established that low external pH combined with unaltered cytoplasmic pH contributes to the protonmotive force which drives flagellar rotation (Khan & Macnab, 1980;Polen et al, 2003). However, weak organic acids that depress the cytoplasmic pH can decrease the proton-motive force and impair the mechanochemical reaction cycle of the flagellar motor but not the actual torque-generation step (Minamino et al, 2003;Nakamura et al, 2009). Taken together, these findings suggest a complex regulation of flagella and flagella-related genes related to different aspects of flagellar structure and function.…”

Section: Discussionmentioning

confidence: 99%

Acid-stress-induced changes in enterohaemorrhagic Escherichia coli O157 : H7 virulence

et al. 2009

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Enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 is naturally exposed to a wide variety of stresses including gastric acid shock, and yet little is known about how this stress influences virulence. This study investigated the impact of acid stress on several critical virulence properties including survival, host adhesion, Shiga toxin production, motility and induction of host-cell apoptosis. Several acid-stress protocols with relevance for gastric passage as well as external environmental exposure were included. Acute acid stress at pH 3 preceded by acid adaptation at pH 5 significantly enhanced the adhesion of surviving organisms to epithelial cells and bacterial induction of host-cell apoptosis. Motility was also significantly increased after acute acid stress. Interestingly, neither secreted nor periplasmic levels of Shiga toxin were affected by acid shock. Pretreatment of bacteria with erythromycin eliminated the acid-induced adhesion enhancement, suggesting that de novo protein synthesis was required for the enhanced adhesion of acidshocked organisms. DNA microarray was used to analyse the transcriptome of an EHEC O157 : H7 strain exposed to three different acid-stress treatments. Expression profiles of acidstressed EHEC revealed significant changes in virulence factors associated with adhesion, motility and type III secretion. These results document profound changes in the virulence properties of EHEC O157 : H7 after acid stress, provide a comprehensive genetic analysis to substantiate these changes and suggest strategies that this pathogen may use during gastric passage and colonization in the human gastrointestinal tract.

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“…This has been done using varying viscous load (10)(11)(12)(13)(14) or external torque (15,16) to control the speed. Fully energized motors with a full complement of stators, driven by H + (10) or Na + (11,12), show the same characteristic torque-speed curve.…”

mentioning

confidence: 99%

Mechanism and kinetics of a sodium-driven bacterial flagellar motor

Sowa

Pilizota

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

104

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The bacterial flagellar motor is a large rotary molecular machine that propels swimming bacteria, powered by a transmembrane electrochemical potential difference. It consists of an ∼50-nm rotor and up to ∼10 independent stators anchored to the cell wall. We measured torque-speed relationships of single-stator motors under 25 different combinations of electrical and chemical potential. All 25 torque-speed curves had the same concave-down shape as fully energized wild-type motors, and each stator passes at least 37 ± 2 ions per revolution. We used the results to explore the 25-dimensional parameter space of generalized kinetic models for the motor mechanism, finding 830 parameter sets consistent with the data. Analysis of these sets showed that the motor mechanism has a "powerstroke" in either ion binding or transit; ion transit is channel-like rather than carrier-like; and the rate-limiting step in the motor cycle is ion binding at low concentration, ion transit, or release at high concentration. where V m is the membrane voltage and Δμ = k B T ln(C in /C out ) and q are the transmembrane chemical potential difference and charge of the ions, respectively, with C in and C out the internal and external ion concentrations. In most species the primary form of biological free energy is the proton-motive force (PMF), the IMF for H + ions (1). Physiological PMF is typically in the range −150 mV to −200 mV, with the inside electrically negative and slightly alkaline relative to the outside. Some organisms use sodium-motive force (SMF) to drive numerous cellular processes, such as bacterial motility (2), ATP synthesis (3), and active membrane transport (4). Arguably the most important process driven by IMF is ATP synthesis, which generates cellular ATP by forced rotation of the F 1 part of F 1 F O ATP-synthase. F 1 is mechanically coupled to and rotated by F O , which like the bacterial flagellar motor (BFM) is an ion-driven rotary motor. Understanding the mechanism of these and other ion-driven molecular machines is a fundamental challenge in cellular energetics and biophysics. The BFM (Fig. 1A) is a rotary molecular machine that propels many species of swimming bacteria. It couples ion flow, for example protons (H + ) in Escherichia coli or sodium ions (Na + ) in Vibrio alginolyticus, to the rotation of extracellular helical flagellar filaments at hundreds of revolutions per second (Hz) (2, 5, 6). Torque is generated by interactions between stator complexes (containing the proteins MotA and MotB in E. coli and PomA and PomB in V. alginolyticus) and the rotor protein FliG (7). In E. coli, each motor can be powered by any number between 1 and at least 11 functionally independent stators (8), which exchange with a membrane-bound pool of "spare" stators on a timescale of minutes (9).The most important biophysical method for studying the torque-generating mechanism of the BFM has been to measure its torque-speed curves. This has been done using varying viscous load (10-14) or external torque (15, 16) to control the speed. Fu...

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Effect of Intracellular pH on the Torque–Speed Relationship of Bacterial Proton-Driven Flagellar Motor

Cited by 73 publications

References 33 publications

Evidence for symmetry in the elementary process of bidirectional torque generation by the bacterial flagellar motor

Evidence for symmetry in the elementary process of bidirectional torque generation by the bacterial flagellar motor

Acid-stress-induced changes in enterohaemorrhagic Escherichia coli O157 : H7 virulence

Mechanism and kinetics of a sodium-driven bacterial flagellar motor

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